The H,K-ATPase was identified as the primary proton secretory pathway in the gastric parietal cell and is the pharmacological target of agents suppressing acid secretion. Recently, we identified a second acid secretory protein expressed in the parietal cell, the vacuolar H-ATPase (V-type ATPase). The aim of the present study was to further characterize H-ATPase activation by modulations in extracellular calcium via the calcium sensing receptor (CaSR). Isolated gastric glands were loaded with the pH indicator dye BCECF-AM [2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester] to measure intracellular pH. Experiments were conducted in the absence of sodium and potassium to monitor H-ATPase-specific transport activity. CaSR was activated with the calcimimetic R568 (400 nM) and/or by modulations in extracellular Ca. Elevation in calcium concentrations increased proton extrusion from the gastric parietal cell. Allosteric modification of the CaSR via R568 and calcium increased vacuolar H-ATPase activity significantly (ΔpH/min = 0.001 ± 0.001, ΔpH/min = 0.033 ± 0.004, ΔpH/min = 0.051 ± 0.005). Carbachol significantly suppressed calcium-induced gastric acid secretion via the H-ATPase under sodium- and potassium-free conditions. We conclude that the V-type H-ATPase is tightly linked to CaSR activation. We observed that proton pump inhibitor (PPI) exposure does not modulate H-ATPase activity. This elevated blood calcium activation of the H-ATPase could provide an explanation for recurrent reflux symptoms while taking a PPI therapy. NEW & NOTEWORTHY This study emphasizes the role of the H-ATPase in acid secretion. We further demonstrate the modification of this proton excretion pathway by extracellular calcium and the activation of the calcium sensing receptor CaSR. The novelty of this paper is based on the modulation of the H-ATPase via both extracellular Ca (activation) and the classical secretagogues histamine and carbachol (inactivation). Both activation and inactivation of this proton pump are independent of PPI modulation.